Mixed Lineage Kinase 3 (MLK3) is a stress-activated MAP Kinase Kinase Kinase (MAP3K) member, and its biological function is still elusive. While dissecting the signaling pathway mediated via MLK3, we observed a robust phosphorylation of a prolyl isomerase Pin1 by MLK3 on Serine138 residue. These results were significant as Pin1 is considered an important oncogene and is overexpressed in almost all types of cancer. The Pin1 specific inhibitors are under development to treat cancer. Remarkably, the phosphorylated Pin1 (i.e. Pin1S138E mutant) was localized in the nucleus, suggesting that this phosphorylation-dependent translocation of Pin1 could have some physiological function. Corroborating our hypothesis, the phosphorylated Pin1 was overexpressed in tumors but not in normal matching tissues and p-Pin1 was necessary for cell cycle progression. Therefore, we ventured to find the targets of p-Pin1 that could promote tumorigenesis. We identified that p-Pin1 (i.e. S138E) was able to up regulate mRNA of Gli1, a transcription factor in the Hedgehog pathway, whose dysregulation has been linked to cancer, including pancreatic cancer. Furthermore, we also observed that mRNA of a chemokine, CXCL5 was downregulated in MLK3 knockout liver, suggesting that MLK3 is necessary for CXCL5 expression. These results were quite intriguing because Gli1 activation/dysregulation has been implicated in pancreatic cancer pathogenesis and also reported that inhibition of Hedgehog pathway overcomes gemcitabine (common drug for pancreatic cancer) resistance in animal model. More recently, CXCL5 and its receptor blockage in pancreatic cancer cell lines was shown to inhibit the pancreatic ductal carcinoma (PDA) cell growth and CXCL5 produced by pancreatic cancer cell lines was able to promote angiogenesis. Our preliminary investigation further revealed that MLK3 activity was regulated by CXCL5 in pancreatic cancer cell lines and the MLK3 activity correlated with the expression levels of CXCL5 in these cell lines. We also observed that in primary human pancreatic tumors, the MLK3 activities directly correlated with the expression levels of Pin1 and Gli1 in tumors. Based on these results, we conceive that the available MLKs inhibitor, CEP-1347/CEP-11004 or in combination with Pin1 inhibitor, should induce cell death in pancreatic cancer cells? Based on our preliminary data, we hypothesize that in pancreatic cancer cells, MLK3 activates Pin1 and its downstream Gli1, leading PDA. Therefore targeting MLK3 or other MLKs along with Pin1 inhibition could abrogate pancreatic tumors. To achieve our goals, we will determine that: (1) activation of Gli1 by MLK3-Pin1 axis promotes pancreatic cell tumorigenesis, (2) disruption of MLK3- Pin1 axis attenuates pancreatic cancer cell tumorigenesis; and (3) we will determine the mechanism of MLK3, Pin1 and Gli1 activation in pancreatic cell tumorigenesis. It is expected that by defining the role of MLK3 in Pin1 and Gli1 activation in pancreatic tumorigenesis, we might control/treat pancreatic cancers by using available inhibitors of this pathway. Interestingly the specific inhibitor of MLK3 family, CEP-1347 has been used in clinical trials for treating neurodegenerative diseases and specific Pin1 inhibitors are under development for cancer treatment. It has also been suggested that MLKs inhibitors might serve as a treatment for specific type of cancer, underscoring our unexpected novel results related to MLK3 role in pancreatic cancer. Taken together, the present study will lead to the identification of new prognostic factors and specific targeted therapies for difficult to treat and lethal pancreatic cancer.